The RGS (Regulators of G-protein Signaling) proteins act to desensitize G protein mediated signal transduction by accelerating the endogenous GTPase activity of activated Gα subunits. RGS proteins have been demonstrated to function as GAPs (GTPase accelerating proteins) for Gαo, Gαi, Gαz and Gαq subtypes of the Gα subunit (Grafstein-Dunn, et al. Mol. Brain Res. 2001, 88:113-123; for a review see Hepler, Trends in Pharmaceutical Sci. 1999, 20:376-382). Therefore, RGS proteins accelerate the turning-off of G-protein coupled receptor GPCR signaling. RGS proteins likely modulate signal transduction of many clinically relevant GPCRs within the CNS. In situ analyses by Gold et al., (Neurosci. 1997, 17(20):8024-37) demonstrated brain specific RGS mRNA expression. Additional in situ evaluation demonstrated overlap of mRNA expression for RGS4, RGS7, and Gαq (Shuey et al., J. Neurochem. 1998, 70:1964-1972).
The first member of the RGS protein family (SST2p) was identified in yeast using a genetically characterized mutant yeast strain (sst) supersensitive to the ligand of the pheromone response pathway (Dohlman et a., Mol Cell Biol. 1996, (9):5194-209). The receptor for this pathway is a G-protein coupled, seven transmembrane receptor (GPCR). The components of this yeast pathway are analogous to those in mammalian GPCR signaling. Subsequently, a dominant mutation was identified in yeast that phenotypically copied a yeast strain deleted for the yeast RGS (SST2p). The mutation also resulted in a supersensitivity to the GPCR ligand, alpha factor, which stimulated the pheromone response pathway in yeast leading to cell cycle arrest. The dominant mutation was identified using genetic studies and molecular biology in yeast (Dohlman et al., (supra), and was due to a G302S mutation in the yeast G protein (Gpa1) that rendered it insensitive to regulation by (RGS) SST2. Gpa1 is homologous to mammalian Gαi proteins. The glycine residue is conserved in mammalian Gα subunits and is contained within the first switch region in the Gα protein. Crystallography studies show that the switch regions of Gαi interacts with RGS4 (Tesmer et al., Science 1997, 278(5345):1907-16). The dominant phenotype (RGS insensitivity) resulting from the G302S mutation in the Gα protein identified in yeast can be transferred to the mammalian Gαq protein (Shuey et al., J. Neurochem. (1998, 70:1964-1972). This work extended the RGS insensitive phenotype of the yeast protein, Gpa1, to mammalian Gαq protein (G188S). The mammalian Gα protein harboring the G to S mutation is able to bind the guanine nucleotide, but is resistant to the GAP activity of RGS proteins.
Since the identification of the first member (yeast protein, SST2) of the RGS protein family in 1996, the impact and biology of the RGS proteins remains to be clarified. RGS proteins are implicated to play a role in brain function, as suggested by region specific expression.
In particular, several GPCRs couple through Gαq to activate second messenger systems (Forse, Crit. Care Med. 2000, 32:524-30; Gudermann et al., Ann. Rev. Neurosci. 1997, 20:399-427) such as PLC, phosphotidyl inositol, Diacyl glycerol, PKC and calcium. Additionally, these messengers can link into MAP kinase pathways to further modulate cellular responses. The Gαq coupled receptors include (but are not limited to) the α1 adrenergic receptor, muscarinic receptors (m1, m3, m5), adrenoreceptors, N-methyl D-aspartate receptors, histamine receptors, serotonin receptors, P2Y, and metabotropic glutamate receptors. Many of these receptors show distinct expression patterns in the brain. The Gαq coupled serotonin receptors (5-HT2A, 5-HT2B, 5-HT2C) are of particular interest because this neurotransmitter system is targeted by several anti-depressant therapeutics.
Of the anti-depressant therapeutics, lithium is the most commonly used treatment for bipolar affective disorder (Jope, Mol. Psychiatry 1999, 4:21-25 and 117-28). Despite its years of usage, the therapeutic mechanism of action of lithium has not been clearly elucidated. Lithium produces a wide spectrum of behavioral and neurochemical effects leading to speculation that its mechanism(s) of action relates to its effects on one or more signaling pathways: G-proteins, IP3, cAMP, wnt, β-catenin, GSK3b, etc. (Williams and Harwood, Trends Pharmaceutical Sci. 2000, 21:61-64; Hedgepath et al., Basic Res. Cardiol. 1997, 92:385-90).
Despite the considerable efforts aimed at elucidating the mechanism of action of therapeutics such as lithium for treating bipolar disorders, there is no clear picture of how such compounds work. An understanding of how compounds such as lithium exert their effects would allow for the design and testing of novel therapeutics that produce a desired therapeutic effect while potentially avoiding adverse side effects. G-protein signaling constitutes an area where additional information could help elucidate specific mechanisms. Accordingly, there is a need for model animal systems which can be used to identify mechanisms of action of RGS blockers and to use as a model for Gαq mediated activity, or discernment of G-protein crosstalk, in receptor function. Establishment of transgenic animals can provide insight into the biological relevance, and potential therapeutic application, for a molecular target.
To this end, transgenic rats that express the RGS insensitive Gαq mutant (G188S) in neuronal tissue were established. These animals were assessed for transgene expression and behaviors to implicate RGS control of Gαq mediated GPCRs of neurological importance.